Pharmaceutical formulations containing 3-(4-cinnamyl-l-piperazinyl) amino derivatives of 3-formylrifamycin sv and 3-formylrifamycin s and a process of their preparation

ABSTRACT

The present invention related to a process of preparation of pharmaceutically acceptable formulations containing as active substance 3-(4-cinnamyl-1-piperazinyl)-amino derivatives of 3-formylrifamycine SV and 3-formylrifamycine S, which possess high activity against Gram-positive and Gram-negative microorganisms, as well as against tuberculous micobacteria (including atypical and rifamycin resistant), and to a method for the preparation of 3-(4-cinnamyl-1-piperazinyl)-amino derivatives of 3-formylrifamycine SV and 3-formylrifamycine S. The method for the preparation of pharmaceutical compositions is readily feasible, and does not require special equipment for its implementation. The process for preparing the compounds is characterized by high yield and purity, using an environmental clean solvent-ethanol and water in the preparation and isolation of substances, and the absence of residual organic solvents in the final product.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Section 371 National Stage Application ofPCT/BG2013/000041, filed Aug. 9, 2013, and published as WO2014026254 onFeb. 20, 2014, which claims priority and benefits of Bulgarian PatentApplication No. 111288, filed Aug. 13, 2012, the entire contents ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention related to a process of preparation ofpharmaceutically acceptable formulations containing as active substance3-(4-cinnamyl-1-piperazinyl)-amino derivatives of 3-formylrifamycine SVand 3-formylrifamycine S, which possess high activity againstGram-positive and Gram-negative microorganisms, as well as againsttuberculous micobacteria (including atypical and rifamycin resistant),and to a method for the preparation of said active compounds. Thepharmaceutical compositions containing active compounds, subject of theinvention, may find use as drugs in medical practice, for theprophylaxis and treatment of various diseases caused by Gram-positiveand Gram-negative microorganisms and by tuberculous mycobacteria(including atypical and rifamycin resistant).

BACKGROUND OF THE INVENTION

In the literature there are described a variety of pharmaceuticalcompositions for the treatment and prophylaxis of the above mentioneddiseases.

It is known the compound 3-(4-methyl-1-piperazinil)-iminomethylrifamycin SV -Rifampicin [U.S. Pat. No. 4,193,020], which ischaracterized by activity against Gram-positive and Gram-negativemicroorganisms and against Mycobacterium tbc.

It is known the compound [U.S. Pat. No. 5,095,108],3-(4-cinnamyl-1-piperazinyl)-iminomethyl rifamycin SV with Formula Ia:

where

R=H and R¹=CH3COO

which has been shown in in vivo tests, higher therapeutic effect incomparison to rifampicin, and considerably longer serum half-life and alow acute toxicity.

It is known the sodium salt of the above compound [U.S. Pat. No.6,476,036], with formula Ib

where

R=Na and R¹=CH3COO

which is characterized by good solubility in water, faster resorptionand better pharmacokinetic properties in in vivo experiments.

Methods are known for the preparation of rifamycin derivatives,including the compound Ia, wherein the 3-formylrifamycin SV, dissolvedin tetrahydrofuran, ethyl acetate, chloroform or a mixture of two ofthese solvents is condensed with an appropriately substitutedN-aminopiperazin, then the reaction mixture was concentrated, and theproduct is recrystallized from an organic solvent, such as acetone orisopropanol [U.S. Pat. No. 4,193,920, U.S. Pat. No. 4,002,752, BG48618]. A disadvantage of the known methods is the relatively low yield(from 55% to 80%), and the necessity of purifying the product byrecrystallization, and further processing of the mother liquors, whichgreatly complicates the process. By applying the method they use largeamounts of organic solvents, which create additional difficulties inregeneration and environment.

A significant disadvantage is the presence of residual solvents(tetrahydrofuran, chloroform, acetone, isopropanol) in the finalproduct, which, due to specific structure of anzamycin molecule can notbe removed even after extensive drying under reduced pressure.Quantities of these solvents, found by gas chromatography, remains abovethe limits set out in EP 5.0/5.4 Known is a method for preparing asodium salt (compound Ib) [U.S. Pat. No. 6,476,036], which comprisesreacting equimolar amounts of a compound Ia with sodium methanolate,ethanolate or isopropanolate in a medium of the corresponding alcohol.The product was isolated by distilling off the solvent under reducedpressure and recrystallization from isopropanol.

The method is not efficient enough, as to give a relatively low yield ofthe product, while there is a need for recrystallization, in addition,in the final product is observed presence of residual organic solventswhose quantities are above the limits set out in EP 5.0/5.4

The above mentioned patent application [U.S. Pat. No. 6,476,036]describes another process for the preparation of the sodium salt, whichis carried out in aqueous medium, adding to the suspension of3-formylrifamycin SV water solution of sodium hydroxide and theresulting aqueous solution was subjected to lyophilisation.

The yield in this case was almost quantitative, but due to strongalkaline reaction medium product contains a number of impurities andneeds recrystallisation from isopropanol, which leads to a significantreduction in yield and the presence of residual solvents in finalproduct A disadvantage of the known methods is the relatively low yield(from 55% to 80%), and the necessity of purifying the product byrecrystallization, and further processing of the mother liquors, whichgreatly complicates the process. Another disadvantage of the method isthe use of organic solvents in large quantities, which createsdifficulties in their regeneration, respectively, while protecting theenvironment. Another major drawback is the presence of residual solvents(tetrahydrofuran, chloroform, acetone, isopropanol) in the finalproduct, which, due to specific structure of anzamycin molecule can notbe removed and after extensive drying under reduced pressure. Quantitiesof these solvents, found by gas chromatography, remains above the limitsset out in EP 5.0/5.4

Due to low solubility of rifamycins in ethyl alcohol the use of ethanolleads to a falling of the finished product as a crystalline residue andits direct isolation by filtration, without the necessity of distillingoff the solvent in advance. At the same time the ethanol makes itdifficult to carrying out the condensation in a conventional manner. Itis generally accepted to a solution of 3-formylrifamycin SV intetrahydrofuran or chloroform to add a solution of N-substitutedaminopiperazin. Using ethyl alcohol as the reaction medium,3-formylrifamycin SV remains undissolved, and the reaction is carriedout in suspension. This leads to incomplete operation of the process andthe presence of unreacted 3-formylrifamycin SV in the final product,which requires purification by recrystallization.

There are not known formulations comprising3-(4-cinnamyl-1-piperazinyl)-amino derivatives of 3-formylrifamycin SV,and 3-formylrifamycin S.

SUMMARY OF THE INVENTION

Taking into account the prior art in this field the task of the presentinvention is to propose pharmaceutical formulations containing as activecompounds 3-(4-cinnamyl-1-piperazinyl)-amino derivatives of3-formylrifamycin SV and 3-formylrifamycin S, with high biologicalactivity.

Another object of the invention is to provide a process for thepreparation of active compounds 3-(4-cinnamyl-1-piperazinyl)-aminoderivatives of 3-formylrifamycin SV and 3-formylrifamycin S, whichpossess high biological activity, providing a very high yield and veryhigh purity, technologically simple in carrying out the process andreduction of its duration.

This object is solved by a pharmaceutical formulations, including theactive substance in a mixture of excipients, depending onphysico-chemical properties of active ingredient, and function of theexcipient.

According to the invention the active substance is a compound,characterized by Formula I and II, as substitutes for R is hydrogen orNa and R1- hydrogen or CH3COO, and its amount in the pharmaceuticalcomposition is from 100 to 600 mg, and the amount of the excipients isfrom 1.5% to 25% based on the total weight of the mixture.

As active substance are used new rifamycin derivatives with formulas Ic,Id, IIa, IIb, IIc and IId, and compounds of formula Ia and Ib.

According to a preferred embodiment of the invention the amount ofactive substance is 150 to 300 mg.

According to one embodiment of the present invention as excipients areused microcrystalline cellulose, corn starch, sodium stearyl fumarate,magnesium stearate.

According to a preferred embodiment of the invention active substancefrom the group of the said active compounds is mixed directly (dryblending) with the excipients and the resulting mixture is filled intocapsules, then the capsules are packaged in a suitable way. The presentinvention is also achieved by a method for the preparation of novelrifamycin derivative, which is carried out by reaction ofN1-cinnamyl-N4-aminopiperazin and 3-formylrifamycin SV, wherein to asolution of aminopiperazin was added 3-rifamycin SV, portion wise, insolid form, and after each portion was awaiting its passage intosolution. The reaction is performed in the presence of small amounts ofacetic acid, at a temperature of 20-30° C. for about 2 hours. Product isobtained as a dark red crystalline precipitate, the reaction mixture isdiluted with equal volume of water and after cooling to 5-10° C. wasfiltered and dried to constant weight.

Compound Ib is prepared by reaction of equimolar quantities of3-(4-cinnamyl-1-piperazinyl)-iminomethyl rifamycin SV (1a) with sodiummethanolate in a medium of ethyl alcohol at room temperature. Theresulting solution of the sodium salt of compound Ia is distilled underreduced pressure and a temperature not higher than 60° C., untilcomplete removal of the ethanol, the residue is dissolved in water inthe presence of small amounts of sodium ascorbate, and the resultingsolution was lyophilized.

The yield of the sodium salt of 3-(4-cinnamyl-1-piperazinyl)-iminomethylrifamycin SV (compound Ib) is quantitative; the product has a very highpurity and does not contain any residual organic solvents.

The new compounds of formulas IIa and IIb are prepared by oxidation ofcompounds Ia and Ib with manganese dioxide in an alcoholic medium,wherein the hydroquinone structure passes into quinone.

Compounds of formulas Ic, Id, IIe and IId are prepared by reaction ofcompounds Ia and IIa with a dilute solution of sodium hydroxide orsodium carbonate in aqueous-alcoholic medium, wherein the ester group in25-position of the molecule, is hydrolyzed, to afford the corresponding25-desacetil derivatives in the form of sodium salts (compounds Id andIId).

Upon acidification of the ethanol solution of the latter two compoundswith dilute hydrochloric acid are received compounds Ic and He.

Newly synthesized rifamycin derivatives can be represented by FormulaIc, Id, IIa, IIb, IIc and IId. Those newly compounds have highantibacterial activity, comparable to that of the compounds Ia and Ib.All synthesized compounds were proven by elementary analysis, IR and UVspectra.

Where for

Ic-R=H, R¹=H

Id-R=Na, R¹=H

where for

IIa-R=H, R¹=COCH3

IIb-R=Na, R¹=COCH3

IIe-R=H, R¹-H

IId-R=Na, R¹=H

The yield of the sodium salt of 3-(4-cinnamyl-1-piperazinyl)-iminomethylrifamycin SV (compound Ib) is quantitative; the product has a very highpurity and does not contain any residual organic solvents. The methodaccording to the invention is characterized by the fact that, thatoperations are carried out in sequence and process parameters which givea product of high purity, as there is no need for further purification,and free from residual amounts of organic solvents. The yield was almostquantitative (98% of theory), and the product has a very high purity anddoes not need further purification, which simplifies the process andreduces its duration. Besides high yield and purity, advantage of themethod is the use of environmentally safe solvents in the preparationand isolation of the substance-ethanol and water, and the absence ofresidual organic solvents in the final product. The method is easy toimplement from a technological standpoint and economically advantageous.

The resulting product is suitable for use in the prophylaxis andtreatment of diseases caused by Gram-positive and Gram-negativemicroorganisms as well as for prophylaxis and treatment of diseasescaused by Mycobacterium tuberculosis (including atypical and rifamycinresistant strains), as according to the invention is suitable theproduct to be made in a suitable pharmaceutically acceptableformulation. In most cases, this may include, but are not limited to,hard gelatin capsules at a dose depending on the activity of thecompound from 100 to 600 mg as a single dose, e.g. 150, 300 or 600 mgand corresponding adjuvants, such as microcrystalline cellulose, starch,magnesium stearate, sodium stearyl fumarate and the like.

Suitably the resulting mixture was filled in capsules, which can bepackaged in a suitable manner, for example in aluminum blisters.

PREFERRED EMBODIMENT OF THE INVENTION

Further, the description will be presented examples of the embodiment ofthe pharmaceutical formulations, and examples of obtaining the activecompounds of formulas I and II, and pharmaceutical formulations are notlimited to the examples described and may also be applied suchembodiments, within the parameters outlined above and ratios.

Example 1

Microcrystalline cellulose (5.60 g) and sodium stearyl fumarate (1.40 g)are sieved and dry- mixed with a pre-weighed amount (30.0 g) of theactive ingredient-sodium salt of3-(4-cinnamyl-1-piperazinyl)-iminomethyl rifamycin SV (compound Ib).After a homogenization, mixture comprising 15.1% of microcrystallinecellulose, 3.8% sodium stearyl fumarate and 81.1% of active substance,is filled into a capsule and then be packaged in a suitable manner, e.g.in aluminum/aluminum blisters. Obtained are 180±5 capsules with anaverage weight of the contents˜185±9 mg.

Example 2

Prior sieved starch (6.00 g) and sodium stearyl fumarate (1.00 g) aredry-mixed with a pre-weighed amount (30.0 g) of sodium salt of3-(4-cinnamyl-1-piperazinyl)-iminomethyl rifamycin S (Compound IIb).After a homogenization mixture is obtained with content-16.2% starch,sodium stearyl fumarate 2.7% and 81.1% of the above-described activeagent. The mixture is filled into a capsule and then be packaged in asuitable manner in aluminum/aluminum blisters. Obtained are 180±5capsules with an average weight of the contents˜185±9 mg.

Example 3

Microcrystalline cellulose (3.0 g) and sodium stearyl fumarate (1.0 g)are sieved and dry mixed well with a pre-weighed amount (60.0 g) ofsodium salt of 3-(4-cinnamyl-1-piperazinyl)-iminomethyl-25-desacetoxyrifamycin SV (Compound Id). The resulting homogeneous mixture containingmicrocrystalline cellulose 4.7%, 1.6% sodium stearyl fumarate and as anactive ingredient the sodium salt of3-(4-cinnamyl-1-piperazinyl)-iminomethyl-25-desacetoxy rifamycinSV-93,8%, is filled into capsules, which are packaged in a suitable wayin aluminum/aluminum blisters. Obtained are 180±5 capsules with anaverage weight of the contents˜320 mg±16 mg.

Example 4

Starch (12.50 g) and magnesium stearate (0.75 g) are sieved anddry-mixed well with a pre-weighed amount (37.0 g) of sodium salt of3-(4-cinnamyl-1-piperazinyl)-iminomethyl-25-desacetoxy rifamycin S(Compound IId). The resulting homogeneous mixture consisting of 12.5%starch, magnesium stearate 1.5% and 74% of active substance is filledinto capsules, which are packaged in an appropriate manner. Obtained are180±5 capsules with an average weight of the contents˜320 mg±16 mg.

Similarly, compositions were prepared with all other derivativesdisclosed already in this patent specification.

Example 5

To a solution of 6.3g (0.0289 gM) N1-cinnamyl-N4-amino piperazine in 200ml of ethanol is added 1.2 ml of glacial acetic acid. Under stirring andat 20-30° C. to the ethanol solution are added portion wise 20 g (0.0276gM) 3-formylrifamycin SV after each batch waiting to dissolve. Additiontakes about 30 min and depletion of the starting rifamycin SV ismonitored by thin layer chromatography. Stirring is continued for 2 hwherein there is a formation of a dark red crystalline precipitate. Thereaction mixture is diluted with 200 ml water, stirred for 15 min andcooled to 5° C. The resulting precipitate of3-(4-cinnamyl-1-piperazinyl) iminomethyl rifamycin SV is filtered andwashed with 20 ml water. After drying under vacuum at 70° C. 25 g of adark red precipitate are obtained, representing 98% of the theoreticalyield. The product has purity greater than 98%.

Example 6

To a suspension of 25 g (24.7 g as 100%) of 3-(4-cinnamyl-1-piperazinyl)iminomethyl rifamycin SV (0.0264 gM) in 200 ml of ethanol with stirring4.88 ml of 30% solution of sodium methoxide (0.0264 gM) is added,wherein the suspension almost immediately pass into solution. Thereaction mixture is filtered and the solvent is distilled under vacuumtill complete elimination. To the resulting gummy residue 200 ml ofwater and 1 g of sodium ascorbate is added. The mixture is stirred untilcomplete dissolving, and subjected to lyophilization. Obtained are 25.2g (99.2% of theory) of the sodium salt of 3-(4-cinnamyl-l-piperazinyl)iminomethyl rifamycin SV in 98% purity.

Example 7

To a solution of 5 g of 3-(4-cinnamyl-1-piperazinyl) iminomethylrifamycin SV in 150 ml of ethanol, 3 g of manganese dioxide is added,the reaction mixture was stirred for 4 hours, and by thin layerchromatography is monitored the passage of the hydroquinone into aquinone The reaction mixture is heated to 50-60° C., and filtered, andthe precipitate of Mn02 is washed thoroughly with warmed ethanol. Theresulting solution is distilled under vacuum to near dryness. Theprecipitate is dried in a vacuum oven at 50° C. Obtained are 4.9 g(98.2% of theory) of 3-(4-cinnamyl-1-piperazinyl) iminomethyl rifamycinS. The product is a dark violet crystalline precipitate.

Example 8

To a solution of 5 g of the sodium salt of 3-(4-cinnamyl-1-piperazinyl)iminomethyl rifamycin SV in 150 ml of ethanol 5 g Mn02 is added and themixture is stirred for 4 hours to complete switching into the quinoneform (TLC). The mixture is heated to 50-60° C., filtered and theprecipitate of Mn02 is washed thoroughly with hot ethanol The solutionis distilled to about ⅓ of the original volume. The residue is dried ina vacuum oven at 50° C. Obtained are dark violet crystals of the sodiumsalt of 3-(4-cinnamyl-1-piperazinyl) iminomethyl rifamycin S. Yield 5 g(97.6 of theory).

Example 9

To a solution of 5 g of sodium hydroxide in 100 ml of a 50% aqueousethanol 5 g of 3-(4-cinnamyl-1-piperazinyl) iminomethyl rifamycin SV isadded Almost immediately, the mixture passes into the solution, in whichafter 10 min begins to fall bright red precipitate. The mixture iscooled to 10° C., filtered and the precipitate is dried in a vacuum ovenat 50° C. Obtained are 4.5 g (92% of theory) of the sodium salt of25-desacetyl-3-(4-cinnamyl-1-piperazinyl) iminomethyl rifamycin SV.

Example 10

To a solution of 3.5 g of sodium salt of25-desacetyl-3-(4-cinnamyl-1-piperazinyl) iminomethyl rifamycin SV in100 ml of ethanol under heating to 40° C., 2 ml of hydrochloric aciddiluted in 10 ml water are added. Orange-red solution is obtained, whichcrystallized upon cooling. The received bright orange precipitate of25-desacetyl-3-(4-cinnamyl-1-piperazinyl) iminomethyl rifamycin SV isfiltered and dried in a vacuum oven at 50° C. Yield 3.3 g (96.5% oftheory).

Example 11

The procedure is as in Example 5 but instead of3-(4-cinnamyl-1-piperazinyl) iminomethyl rifamycin SV is used3-(4-cinnamyl-1-piperazinyl) iminomethyl rifamycin S. Obtained is sodiumsalt of 3-(4-cinnamyl-1-piperazinyl) iminomethyl rifamycin S in 90%yield of theory.

Example 12

The product of Example 7 was subjected to the procedure described inExample 6. Prepared is 25-desacetyl-3-(4-cinnamyl-1-piperazinyl)iminomethyl rifamycin S in 95% yield

1. Pharmaceutical formulations comprising a mixture of the activesubstance of a compound from the group3-(4-cinnamyl-1-piperazinyl)-amino derivatives of 3-formylrifamycin SV,and S 3-formylrifamycin and excipients, characterized by the fact thatthe amount of active ingredient per dosage unit is from 100 to 600 mg,and the excipients are present in an amount from 1 to 25% based on thetotal weight of the mixture.
 2. Pharmaceutical compositions in which theactive ingredient is a compound of formula Ia, Ib, Ic and Id

where for: Ia-R=H, R¹=COCH₃; Ib-R=Na, R¹=COCH₃; Ic-R=H, R¹=H; Id-R=Na,R¹=H
 3. Pharmaceutical formulations according to claim 1 characterizedby the fact that the active substance is a compound of formula II

where for: IIa-R=H, R¹=COCH₃; IIb-R=Na, R¹=COCH₃; IIc-R=H, R¹=H;IId-R=Na, R¹=H
 4. Pharmaceutical formulations according to claim 1,characterized by the fact that as auxiliaries are used microcrystallinecellulose, starch.
 5. Pharmaceutical formulations according to claim 1characterized by the fact that some excipients used are slidingexcipients, such as sodium stearyl fumarate, magnesium stearate.
 6. Aprocess for the preparation of 3-(4-cinnamyl-1-piperazinyl)-iminomethylrifamycin SV by reacting 3-formylrifamycin SV withN1-cinnamyl-N4-aminopiperazin, characterized by the fact that to anethanol solution of N1-cinnamyl-N4-aminopiperazin in the presence ofsmall amounts of acetic acid and a temperature of 20-30° C.3-formylrifamycin SV is added apart, the reaction mixture is dilutedwith equal volume of water, filtered and the precipitate is washed withwater.
 7. A process for preparing a sodium salt of the compoundaccording to claim 6, characterized by the fact that the3-(4-cinnamyl-1-piperazinyl) iminomethyl rifamycin SV is reacted with anequimolar amount of sodium methoxide in ethanol, at temperature of20-30° C., followed by distilling off the ethanol, the residue isdissolved in water in the presence of sodium ascorbate, and the aqueoussolution is lyophilized.
 8. Compounds of general formula I,

wherein R can be H or Na, and R1 is H.
 9. Compound according to claim 8,wherein R=H and R¹=H.
 10. Compound according to claim 8, wherein R=Naand R¹=H
 11. Compounds of general formula II

wherein R can be H or Na, and R1 is COCH3 or H
 12. Compound according toclaim 11 wherein R=H and R¹=COCH₃
 13. Compound according to claim 11wherein R=Na and R¹=COCH₃
 14. Compound according to claim 11 wherein R=Hand R¹=H
 15. Compound according to claim 11 wherein R=Na and R¹=H 16.Compound according to claim 8 for use as medicament.
 17. Compoundaccording to claim 8 for use in the treatment of diseases and conditionscaused by Gram-positive or Gram-negative bacteria.
 18. Compoundaccording to claim 8 for use in the treatment of diseases and conditionscaused by tuberculous mycobacterium.
 19. Compound according to claim 8for use in the treatment of tuberculosis including atypical andrifamycin resistant forms of tuberculosis.
 20. Compound according toclaim 11 for use as medicament
 21. Compound according to claim 11 foruse in the treatment of diseases and conditions caused by Gram-positiveor Gram-negative bacteria.
 22. Compound according to claim 11 for use inthe treatment of diseases and conditions caused by tuberculousmycobacterium.
 23. Compound according to claim 11 for use in thetreatment of tuberculosis including atypical and rifamycin resistantforms of tuberculosis.